The present invention relates to infrared absorption spectroscopy of molecules and, in particular, to In-Situ Ultra-Sensitive infrared absorption spectroscopy of biomolecules and biomolecule interactions in real-time with Plasmonic Nanoantennas.
Infrared absorption spectroscopy is a powerful biochemical analysis tool as it extracts detailed molecular structural information in a label-free fashion. Its molecular specificity renders the technique sensitive to the subtle conformational changes exhibited, for example, by proteins in response to a variety of stimuli. Yet, sensitivity limitations and the extremely strong absorption bands of liquid water severely limit infrared spectroscopy in performing kinetic measurements in biomolecules' native, aqueous environments.
Infrared (IR) absorption spectroscopy directly probes the vibrational modes associated with the various molecular bonds in a sample by measuring absorption in the mid-IR spectral region, ˜3-20 μm (3000-600 cm−1. As such, IR spectroscopy measurements are intrinsically endowed with a level of chemical specificity and information content far exceeding most other optical measurement techniques. Fluorescence based measurements rely on an exogenous label for their signal, while refractive index (RI) sensors essentially monitor a non-specific bulk property, mass accumulation. In contrast, signal in IR spectroscopy originates with the most intrinsic part of a sample—its molecular structure. IR measurements can thus be leveraged for automated tissue classification and cancer identification. Most significant is their ability to record the conformational changes of proteins that elucidate the molecular mechanisms of their function. Critically, such measurements do not involve any transfer of mass and are therefore largely inaccessible to other methods.
Despite such advantages, several shortcomings severely limit the application of IR absorption spectroscopy in the measurement of biological samples and their dynamic behavior in real-time. These are concerned with sensitivity and difficulties in sampling in aqueous solutions. Sensitivity is limited as a result of Beer's law, which implies that for the small IR absorption cross sections, in thin samples such as monolayers, absorption signals become prohibitively weak. For measurement in solution, water, though an essential component of most biological processes, presents the major obstacle. Specifically, its OH bending absorption can overwhelm any signal from protein samples. Therefore, special care to limit path lengths to less than 10 μm and protein concentrations of several tens of mg/mL are needed to obtain the high signal-to-noise (SNR) level data required for functional studies when measurements are performed in solution.
This second issue is addressed in part by attenuated total internal reflection (ATR) sampling, which achieves a fixed path length via the evanescent field used to probe the sample. Yet, without any signal amplification it cannot achieve adequate sensitivity. This fundamental limitation can be overcome by leveraging the strong light-matter interaction and sub-wavelength localization enabled by the plasmonic resonances of nano-scale metallic particles, resulting in the phenomena of surface enhanced infrared absorption (SEIRA), in analogy with surface enhanced Raman scattering (SERS). While early SEIRA studies utilized metal island films, prepared by e.g. chemical means or physical vapor deposition, these are stochastic in nature, provided limited enhancement and suffered from significant repeatability issues. In contrast, recent work has shown that explicitly engineered plasmonic nanoantennas with mid-IR resonance offer reliable, 104-105 fold enhancement, occurring at well-defined deterministic locations. To date, however, all such resonant SEIRA measurements have been performed on dry samples, strongly limiting the use of powerful infrared spectroscopy for in-situ studies with live cells, tissues etc. and performing real-time measurements, i.e. biomolecular kinetic interactions important for biology, biochemistry, pharmacology.